ATF4通过调节Parkin减轻棕榈酸诱导的肾小球足细胞凋亡

ATF4 alleviates palmitic acid-induced apoptosis of glomerular podocytes by regulating Parkin

目的:探讨转录激活因子4(activating transcription factor-4,ATF4)调节Parkin在棕榈酸(palmitic acid,PA)诱导的肾小球足细胞凋亡中的作用。方法:SD大鼠20只随机分2组:普通饮食组(n=10)及高脂饮食组(n=10)喂养12周后,采集血清检测甘油三酯(triglyceride,TG)、总胆固醇酯(total cholesterol,TC);Tunel染色检测肾组织细胞凋亡情况;免疫组化、Western blot检测肾组织Cleaved-caspase3和ATF4、Parkin蛋白的表达。在体外实验中,采用PA刺激足细胞,或用质粒转染过表达Parkin或用腺病毒敲低ATF4表达,Western blot检测足细胞凋亡相关蛋白Cleaved-caspase3表达水平,流式细胞术检测足细胞的凋亡率。结果:高脂饮食组大鼠TG和TC的表达水平(2.700±0.565、2.620±0.565)分别与普通饮食组(0.780±0.165、1.540±0.377)相比明显增加(t=-7.987,P=0.000;t=-3.895,P=0.003)。肾组织凋亡相关蛋白Cleaved-caspase3、Parkin蛋白和ATF4蛋白表达水平,高脂饮食组(2.257±0.326、1.587±0.162、1.851±0.347)较普通饮食组(1.000±0.190、1.000±0.323、1.000±0.186)明显增加(P=0.000,P=0.003,P=0.000)。采用不同浓度PA刺激发现,足细胞ATF4、Parkin及Cleaved-caspase3的表达量150μmol/L PA组(1.689±0.235、1.529±0.057、1.773±0.186)、300μmol/L PA组(1.702±0.228、2.047±0.086、2.282±0.155)较对照组(1.000±0.030、1.000±0.066、1.000±0.039)明显增加(P=0.002,P=0.002;P=0.000,P=0.000;P=0.000,P=0.000)。采用Parkin过表达质粒转染足细胞后,与对照组(1.000±0.253)相比,Parkin过表达组的Parkin表达(105.242±24.108)明显升高(P=0.000)。进一步予以PA刺激,PA+Parkin过表达组足细胞[1.943±0.200,(9.590±0.418)%]较PA+空载质粒组[3.398±0.164,(15.140±1.227)%]其Cleaved-caspase3明显减少(P=0.000),细胞凋亡率明显降低(P=0.000)。进行腺病毒转染敲低ATF4后,与对照组(1.000±0.112、1.000±0.104、1.000±0.279)相比,shATF4 3组ATF4蛋白表达(0.175±0.064)、Parkin蛋白表达(0.518±0.130)明显减少(P=0.000,P=0.004),Cleavedcaspase3表达(2.305±0.150)明显增加(P=0.000)。细胞凋亡率shATF4组[(14.230±0.278)%]较对照组[(7.020±0.773)%]明显增加(P=0.000)。PA刺激已敲低ATF的足细胞,Cleaved-caspase3及细胞凋亡率相比PA+shRNA组[1.926±0.215,(20.943±4.194)%],PA+shATF4组[2.483±0.114,(46.643±10.675)%]明显增加(P=0.007,P=0.018)。结论:ATF4可以调节Parkin来减轻棕榈酸诱导的肾小球足细胞凋亡。

Objective:To investigate the role of activating transcription factor-4(ATF4)in palmitic acid(PA)-induced apoptosis of glomerular podocytes by regulating Parkin.Methods:Twenty Sprague Dawley rats were randomly divided into two groups:normal diet group with 10 rats and high-fat diet group with 10 rats.After 12 weeks of feeding,serum was collected for measurement of triglyceride(TG)and total cholesterol(TC)levels.Cell apoptosis in renal tissues was evaluated by Tunel staining.Immunohistochemical method and Western blot were used to measure the protein expression of cleaved cysteinyl aspartate specific proteinase-3(Cleaved caspase-3),ATF4,and Parkin in renal tissues.PA was used to stimulate glomerular podocytes in vitro,plasmids were trans-fected with overexpressed Parkin,or adenovirus was used to knock down the expression of ATF4.Western blot was used to measure the expression of Cleaved caspase-3,and flow cytom-etry was used to determine the apoptosis rate of glomerular podocytes.Results:The high-fat diet group had significantly higher expression of TG and TC compared with the normal diet group(2.700±0.565 vs.0.780±0.165,t=-7.987,P=0.000;2.620±0.565 vs.1.540±0.377,t=-3.895,P=0.003).The protein expression of Cleaved caspase-3,Parkin,and ATF4 were significantly higher in the high-fat diet group than in the normal diet group(2.257±0.326 vs.1.000±0.190,P=0.000;1.587±0.162 vs.1.000±0.323,P=0.003;1.851±0.347 vs.1.000±0.186,P=0.000).After being stimu-lated with different concentrations of PA,compared with the control group,the expression of ATF4,Parkin,and Cleaved caspase-3 in podocytes was significantly increased in the 150μmol/L PA group(1.689±0.235 vs.1.000±0.030,P=0.002;1.529±0.057 vs.1.000±0.066,P=0.000;1.773±0.186 vs.1.000±0.039,P=0.000)and in the 300μmol/L PA group(1.702±0.228 vs.1.000±0.030,P=0.002;2.047±0.086 vs.1.000±0.066,P=0.000;2.282±0.155 vs.1.000±0.039,P=0.000).After the Parkin overexpression plas-mid was transfected into podocytes,the expression of Parkin in the Parkin overexpression group was significantly increased compared with the control group(105.242±24.108 vs.1.000±0.253,P=0.000).After stimulation with PA,the expression of Cleaved caspase-3 in podocytes and PA-induced apoptosis rate in PA+Parkin overexpression group were significantly reduced compared with the PA+empty plasmid group[1.943±0.200 vs.3.398±0.164,P=0.000;(9.590±0.418)%vs.(15.140±1.227)%,P=0.000].After adenovirus transfection and knockdown of ATF4,the protein expression of ATF4 and Parkin in the shATF4 group was significantly reduced and the expression of Cleaved caspase-3 was significantly increased compared with the control group(0.175±0.064 vs.1.000±0.112,P=0.000;0.518±0.130 vs.1.000±0.104,P=0.004;2.305±0.150 vs.1.000±0.279,P=0.000).The cell apoptosis rate of podocytes was significantly higher in the shATF4 group than in the control group[(14.230±0.278)%vs.(7.020±0.773)%,P=0.000].After podocytes were stimulated with ATF knockdown,the expression of Cleaved caspase-3 and apoptosis rate were significantly higher in the PA+shATF4 group than in the PA+shRNA group[2.483±0.114 vs.1.926±0.215,P=0.007;(46.643±10.675)%vs.(20.943±4.194)%,P=0.018].Conclusion:ATF4 can alleviate PA-induced apoptosis of glomerular podocytes by regulating Parkin。